Antibody immunologically reactive with serine protease EOS

ABSTRACT

Here we describe the molecular identification of a cDNA encoding a novel serine protease we have termed protease EOS. The deduced amino acid sequence, and it alignment with other well-characterized serine proteases indicates that it is a member of the S1 serine protease family. We have found that the protease EOS mRNA is expressed in platelets and leukocytes and more specifically eosinophils. Although this protease is abundantly expressed in ovary, retina and stomach, where it may perform important functions, its expression in platelets and certain cells of the immune system suggests that it may play roles in thrombosis and in the immune process. Enzymatically active protease EOS is amenable to further biochemical analyses for the identification of physiological substrates and specific modulators.

This is a Divisional of application Ser. No.: 09/387,375, filed Aug. 31,1999 now U.S. Pat. No. 6,485,957.

BACKGROUND OF THE INVENTION

Members of the trypsin/chymotrypsin-like (S1) serine protease familyplay pivotal roles in a multitude of diverse physiological processes,including digestive processes and regulatory amplification cascadesthrough the proteolytic activation of inactive zymogen precursors. Inmany instances protease substrates within these cascades are themselvesthe inactive form, or zymogen, of a “downstream” serine protease.Well-known examples of serine protease-mediated regulation include bloodcoagulation, (Davie, et al (1991). Biochemistry 30:10363-70), kininformation (Proud and Kaplan (1988). Ann Rev Immunol 6: 49-83) and thecomplement system (Reid and Porter (1981). Ann Rev Biochemistry50:433-464). Although these proteolytic pathways have been known forsometime, it is likely that the discovery of novel serine protease genesand their products will enhance our understanding of regulation withinthese existing cascades, and lead to the elucidation of entirely novelprotease networks.

Differentiated blood cells express an assortment of proteases that arelikely to play specific roles in various pathological states. Althoughgranzymes from cytotoxic T cells and natural killer (NK) cells (Smyth etal. (1996). J. Leukocyte Biol. 60:555-562), elastase and collagenasesfrom neutrophils (Simon (1993). Agents Actions Suppl. 42:27-37) andchymase and tryptase from mast cells (Caughey (1995). Clin. AllergyImmunol. 6:305-29; Katunuma and Kido (1988). J. Cell. Biochem.38:291-301) are currently under investigation, their roles inpathophysiological processes are only now being elucidated. In contrast,the proteases from eosinophils have not been characterized and are onlycurrently being molecularly identified. Understanding the physiologicalroles these eosinophil proteases play will lead to a betterunderstanding of eosinophil function in health and diseased states(Abu-Ghazaleh et al. (1992). Immunol. Ser. 57:137-67; Gleich (1996).Allergol. Int. 45:35-44; Gleich et al. (1993). Annu. Rev. Med.44:85-101).

Proteases are used in non-natural environments for various commercialpurposes including laundry detergents, food processing, fabricprocessing, and skin care products. In laundry detergents, the proteaseis employed to break down organic, poorly soluble compounds to moresoluble forms that can be more easily dissolved in detergent and water.In this capacity the protease acts as a “stain remover.” Examples offood processing include tenderizing meats and producing cheese.Proteases are used in fabric processing, for example, to treat wool inorder prevent fabric shrinkage. Proteases may be included in skin careproducts to remove scales on the skin surface that build up due to animbalance in the rate of desquamation. Common proteases used in some ofthese applications are derived from prokaryotic or eukaryotic cells thatare easily grown for industrial manufacture of their enzymes, forexample a common species used is Bacillus as described in U.S. Pat. No.5,217,878. Alternatively, U.S. Pat. No. 5,278,062 describes serineproteases isolated from a fungus, Tritirachium album, for use in laundrydetergent compositions. Unfortunately use of some proteases is limitedby their potential to cause allergic reactions in sensitive individualsor by reduced efficiency when used in a non-natural environment. It isanticipated that protease proteins derived from non-human sources wouldbe more likely to induce an immune response in a sensitive individual.Because of these limitations, there is a need for alternative proteasesthat are less immunogenic to sensitive individuals and/or providesefficient proteolytic activity in a non-natural environment. The adventof recombinant technology allows expression of any species' proteins ina host suitable for industrial manufacture.

SUMMARY OF THE INVENTION

Here we describe the molecular identification of a cDNA encoding a novelserine protease we have termed protease EOS. The protease EOS cDNAsequence predicts a preproEOS polypeptide of 284 amino acids, and itsalignment with other well-characterized serine proteases clearlyindicates that it is a member of the S1 serine protease family.

Enzymatically active protease EOS is amenable to further biochemicalanalyses for the identification of physiological substrates and specificmodulators. Modulators identified in the chromogenic assay disclosedherein are potentially useful as therapeutic agents in the treatment ofdiseases associated with platelet function or elevated eosinophil countssuch as in, but not limited to, bronchial asthma and complicationsarising from hypereosinophilia. In addition, expression of protease EOSin the ovary, retina and stomach suggests that modulators of proteaseEOS function could be used to treat disorders effecting these tissues.Purified protease EOS can be manufactured as a component for use incommercial products including laundry detergents, stain-removingsolutions, and skin care products.

The recombinant DNA molecules coding for EOS, and portions thereof, areuseful for isolating homologues of the DNA molecules, identifying andisolating genomic equivalents of the DNA molecules, and identifying,detecting or isolating mutant forms of the DNA molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—The nucleotide (SEQ.ID.NO.::1) and amino acid sequence(SEQ.ID.NO.: 7) of the novel protease EOS cDNA is shown.

FIG. 2—The phylogenetic tree of the protease EOS amino acid sequencerelative to other S1 serine proteases is shown.

FIG. 3—PCR-based tissue distribution indicates that the protease EOSmRNA is restricted. Autoradiograms of gels are shown with the positionof the EOS specific PCR product (EOS), as detected by the hybridizationof a labeled nested probe, which was resolved following electrophoresisfrom the free probe (F.P.). The cDNA libraries of tissues and cell linesanalyzed are as indicated.

FIG. 4—The nucleotide and amino acid sequences of the protease EOScatalytic domain in the zymogen activation construct is shown.

FIG. 5—Polyacrylamide gel and Western blot analyses of the recombinantprotease PFEK-protease EOS-HA6XHIS. Shown is the polyacrylamide gelcontaining samples of the novel serine protease PFEK-proteaseEOS-HA6XHIS stained with Coomassie Brilliant Blue (Leftmost 1, 2. ). Therelative molecular masses are indicated by the positions of proteinstandards (M). In the indicated lanes, the purified zymogen was eitheruntreated (−) or digested with EK (+) which was used to cleave andactivate the zymogen into its active form. Western blot of the gel,probed with the anti-FLAG MoAb M2, is also shown (rightmost 1). Thisdemonstrates the quantitative cleavage of the expressed and purifiedzymogen to generate the processed and activated protease.

FIG. 6—Functional amidolytic activities of the recombinant proteaseEOS-HA6XHIS expressed, purified and activated from the activationconstruct were determined using chromogenic substrates.

DETAILED DESCRIPTION

Definitions

The term “protein domain” as used herein refers to a region of a proteinthat can fold into a stable three-dimensional structure independent tothe rest of the protein. This structure may maintain a specific functionassociated with the domain's function within the protein includingenzymatic activity, creation of a recognition motif for anothermolecule, or provide necessary structural components for a protein toexist in a particular environment. Protein domains are usuallyevolutionarily conserved regions of proteins, both within a proteinsuperfamily and within other protein superfamilies that perform similarfunctions.

The term “protein superfamily” as used herein refers to proteins whoseevolutionary relationship may not be entirely established or may bedistant by accepted phylogenetic standards, but show similar threedimensional structure or display unique consensus of critical aminoacids. The term “protein family” as used herein refers to proteins whoseevolutionary relationship has been established by accepted phylogenicstandards.

The term “fusion protein” as used herein refers to protein constructsthat are the result of combining multiple protein domains or linkerregions for the purpose of gaining function of the combined functions ofthe domains or linker regions. This is most often accomplished bymolecular cloning of the nucleotide sequences to result in the creationof a new polynucleotide sequence that codes for the desired protein.Alternatively, creation of a fusion protein may be accomplished bychemically joining two proteins together.

The term “linker region” or “linker domain” or similar such descriptiveterms as used herein refers to stretches of polynucleotide orpolypeptide sequence that are used in the construction of a cloningvector or fusion protein. Functions of a linker region can includeintroduction of cloning sites into the nucleotide sequence, introductionof a flexible component or space-creating region between two proteindomains, or creation of an affinity tag for specific moleculeinteraction. A linker region may be introduced into a fusion proteinwithout a specific purpose, but results from choices made duringcloning.

The term “pre-sequence” as used herein refers to a nucleotide sequencethat encodes a secretion signal amino acid sequence. A wide variety ofsuch secretion signal sequences are known to those skilled in the art,and are suitable for use in the present invention. Examples of suitablepre-sequences include, but are not limited to, prolactinFLAG,trypsinogen, and chymoFLAG.

The term “pro-sequence” as used herein refers to a nucleotide sequencethat encodes a cleavage site for a restriction protease. A wide varietyof cleavage sites for restriction proteases are known to those skilledin the art, and are suitable for use in the present invention. Examplesof suitable pro-sequences include, but are not limited to, EK, FXa, andthrombin.

The term “cloning site” or “polycloning site” as used herein refers to aregion of the nucleotide sequence contained within a cloning vector orengineered within a fusion protein that has one or more availablerestriction endonuclease consensus sequences. The use of a correctlychosen restriction endonuclease results in the ability to isolate adesired nucleotide sequence that codes for an in-frame sequence relativeto a start codon that yields a desirable protein product aftertranscription and translation. These nucleotide sequences can then beintroduced into other cloning vectors, used create novel fusionproteins, or used to introduce specific site-directed mutations. It iswell known by those in the art that cloning sites can be engineered at adesired location by silent mutations, conserved mutation, orintroduction of a linker region that contains desired restriction enzymeconsensus sequences. It is also well known by those in the art that theprecise location of a cloning site can be flexible so long as thedesired function of the protein or fragment thereof being cloned ismaintained.

The term “tag” as used herein refers to a nucleotide sequence thatencodes an amino acid sequence that facilitates isolation, purificationor detection of a fusion protein containing the tag. A wide variety ofsuch tags are known to those skilled in the art, and are suitable foruse in the present invention. Suitable tags include, but are not limitedto, HA-tag, His-tag, biotin, avidin, and antibody binding sites.

As used herein, “expression vectors” are defined herein as DNA sequencesthat are required for the transcription of cloned copies of genes andthe translation of their mRNAs in an appropriate host. Such vectors canbe used to express eukaryotic genes in a variety of hosts such asbacteria including E. coli, blue-green algae, plant cells, insect cells,fungal cells including yeast cells, and animal cells.

The term “catalytic domain cassette” as used herein refers to anucleotide sequence that encodes an amino acid sequence encoding atleast the catalytic domain of the serine protease of interest. A widevariety of protease catalytic domains may be inserted into theexpression vectors of the present invention, including those presentlyknown to those skilled in the art, as well as those not yet having anisolated nucleotide sequence encodes it, once the nucleotide sequence isisolated.

As used herein, a “functional derivative” of the nucleotide sequence,vector, or polypeptide possesses a biological activity (eitherfunctional or structural) that is substantially similar to theproperties described herein. The term “functional derivatives” isintended to include the “fragments,” “variants,” “degenerate variants,”“analogs” and “homologues” of the nucleotide sequence, vector, orpolypeptide. The term “fragment” is meant to refer to any nucleotidesequence, vector, or polypeptide subset of the modules described as preand pro sequences used for the activation of expressed zymogenprecursors. The term “variant” is meant to refer to a nucleotide oramino acid sequence that is substantially similar in structure andfunction to either the entire nucleic acid sequence or encoded proteinor to a fragment thereof. A nucleic acid or amino acid sequence is“substantially similar” to another if both molecules have similarstructural characteristics or if both molecules possess similarbiological properties. Therefore, if the two molecules possesssubstantially similar activity, they are considered to be variants evenif the structure of one of the molecules is not found in the other oreven if the two amino acid sequences are not identical. The term“analog” refers to a protein molecule that is substantially similar infunction to another related protein.

Herein we describe a serine protease isolated from eosinophil cellstermed EOS. The protease EOS deduced amino acid sequence is most similarto the cloned serine proteases prostasin (Yu et al. (1996). Genomics32:334-40) and tryptase (Miller et al. (1990). J. Clin. Invest.86:864-700). Tryptase, which is produced abundantly in mast cells, hasbeen implicated in asthmatic inflammation and obstruction (Johnson etal. (1997). Eur. Respir. J. 10:38-43). Additional homology searches ofthe Genbank database with the protease EOS nucleotide sequence revealedhomology with non-contiguous regions of the human cosmid clone (407D8,Genbank accession #AC005570), which maps to chromosome 16p13.3. Assemblyof a continuous nucleic acid sequence from the proposed intron/exonjunctions described in the Genbank accession #AC005570 annotationproduces a nucleic acid sequence that is shorter and alsonon-contiguous, and thus substantially different from, protease EOS ofthe present invention.

Thus, it is likely that the exons delineated in the Genbank accession#AC005570 annotation are incorrect. Therefore, protease EOS of thepresent invention represents a previously undescribed protease. The useof the previously undescribed sequence of the present inventionindicates that chromosome 16p13.3 is the correct the position of theprotease EOS gene. Interestingly, the gene encoding prostasin has beenlocalized on chromosome 16p11.2 (Yu et al. (1996). Genomics 32:334-40)while several tryptase genes are even clustered within the samechromosome 16p13.3 genomic interval (Pallaoro et al. (1999). J. Biol.Chem. 274:3355-3362), and are consequently co-localized with the genefor protease EOS. Recent genetic data links determinants ofsusceptibility to asthma on chromosome 16 in some populations (Danielset al. (1996). Nature (London) 383:247-253). Eosinophilia, a conditioncharacterized by elevated circulating eosinophils, is associated withnumerous allergic states including bronchial asthma (Gleich (1996).Allergol. Int. 45:35-44). Therefore protease EOS, or manipulation ofthis enzyme by chemical modulators, may be useful for treatment ofimmune processes involving eosinophils, with asthma being one importantexample.

Proteases are used in non-natural environments for various commercialpurposes including laundry detergents, food processing, fabricprocessing, and skin care products. In laundry detergents, the proteaseis employed to break down organic, poorly soluble compounds to moresoluble forms that can be more easily dissolved in detergent and water.In this capacity the protease acts as a “stain remover.” Examples offood processing include tenderizing meats and producing cheese.Proteases are used in fabric processing, for example, to treat wool inorder prevent fabric shrinkage. Proteases may be included in skin careproducts to remove scales on the skin surface that build up due to animbalance in the rate of desquamation. Unfortunately use of someproteases is limited by their potential to cause allergic reactions insensitive individuals or by reduced efficiency when used in anon-natural environment. Because of these limitations, there is a needfor alternative proteases that are less immunogenic to sensitiveindividuals and/or provides efficient proteolytic activity in anon-natural environment. Because protease EOS is derived from a humanhost, it is less likely to induce an allergic reaction in sensitiveindividuals, and therefore protease EOS may also be useful forformulation of compositions for laundry detergents and skin careproducts.

The present invention relates to DNA encoding the serine protease EOSthat was identified from an eosinophil library, constructed using poly ARNA isolated from pooled diseased eosinophils obtained from allergicasthmatic individuals. The protease EOS as used herein, refers to theencoded protein product which can specifically function as a protease.

The complete amino acid sequence of protease EOS was not previouslyknown, nor was the complete nucleotide sequence encoding protease EOSknown. This is the first reported cloning of a full length DNA moleculeencoding protease EOS. Based on mRNA distribution, it is predicted thata restricted number of tissues and cell types will contain the describedprotease. Vertebrate cells capable of producing protease EOS include,but are not limited to eosinophils isolated from blood. Other tissuetypes may be human spleen, retina, spinal cord and ovary.

Other cells and cell lines may also be suitable for use to isolate theprotease EOS cDNA. Selection of suitable cells may be done by screeningfor protease EOS proteolytic activity in conditioned cell media. Celltypes that possess EOS proteolytic activity in this assay may besuitable for the isolation of the protease EOS DNA or mRNA.

Any of a variety of procedures known in the art may be used tomolecularly clone protease EOS DNA. These methods include, but are notlimited to, direct functional expression of protease genes following theconstruction of a protease EOS-containing cDNA library in an appropriateexpression vector system. Another method is to screen proteaseEOS-containing cDNA library constructed in a bacteriophage or plasmidshuttle vector with a labeled oligonucleotide probe designed from theamino acid sequence of the protease EOS DNA. An additional methodconsists of screening a protease EOS-containing cDNA library constructedin a bacteriophage or plasmid shuttle vector with a partial cDNAencoding the protease EOS protein. This partial cDNA is obtained by thespecific polymerase chain reaction (PCR) amplification of protease EOSDNA fragments through the design of degenerate oligonucleotide primersfrom the amino acid sequence of the purified protease EOS protein.Expressed sequence tags (EST)s, identified through homology searching ofnucleic acid databases (Altschul et al. (1990). J. Mol. Biol.215:403-10; Pearson and Lipman (1988). Proc. Natl. Acad. Sci. U.S.A.85:2444-8), are also available for this purpose. This particularprotease is a member of a multigene family containing highly conservedresidues and motifs. Thus, cDNA library screening under reducedstringency to identify related but non-identical homologous cDNAs ispossible. More recently, direct PCR using degenerate oligonucleotides ofcDNA reverse transcribed from RNA of a given cell type, has been afruitful approach to isolate novel related cDNAs of interest.Alternatively, the full-length cDNA sequence once published, may beobtained by the specific PCR amplification, through the design ofmatching oligonucleotide primers flanking the entire coding sequence.

Another method is to isolate RNA from protease EOS-producing cells andtranslate the RNA into protein via an in vitro or an in vivo translationsystem. The translation of the RNA into a protein will result in theproduction of at least a portion of the protease EOS protein that can beidentified by, for example, immunological reactivity with ananti-protease EOS antibody. Should the entire catalytic domain betranslated, functional proteolytic activity of the EOS protein couldconceivably be used to identify RNA fractions containing the proteaseEOS mRNA. In this method, pools of RNA isolated from proteaseEOS-producing cells can be analyzed for the presence of an RNA thatencodes at least a portion of the EOS protein. Further fractionation ofthe RNA pool can be done to purify the protease EOS RNA fromnon-protease EOS RNA. The peptide or protein produced by this method maybe analyzed to provide amino acid sequences, which in turn may be usedto provide primers for production of protease EOS cDNA. Similarly, RNAused for translation can be analyzed to provide nucleotide sequences andmay be used to produce probes for the production of the protease EOScDNA. This method is known in the art and can be found in, for example,(Maniatis et al. (1989). 1-1626).

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cells or celltypes, may be useful for isolating protease EOS-encoding DNA. Othertypes of libraries include, but are not limited to, cDNA librariesderived from other cells, from non-human organisms, and genomic DNAlibraries that include YAC (yeast artificial chromosome) and cosmidlibraries.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have EOSproteolytic activity. The selection of cells or cell lines for use inpreparing a cDNA library to isolate the protease EOS cDNA may be done byfirst measuring cell associated EOS proteolytic activity using themeasurement of protease EOS-associated biological activity or a EOSspecific immunological reactivity.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in (Maniatis et al. (1989). 1-1626).

It is also readily apparent to those skilled in the art that DNAencoding protease EOS may also be isolated from a suitable genomic DNAlibrary. Construction of genomic DNA libraries can be performed bystandard techniques well known in the art. Well known genomic DNAlibrary construction techniques can be found in (Maniatis et al. (1989).1-1626).

In order to clone the protease EOS gene by the above methods, the aminoacid sequence of protease EOS may be necessary. To accomplish this, theprotease EOS protein may be purified and partial amino acid sequencedetermined by automated sequencers. It is not necessary to determine theentire amino acid sequence, but the linear sequence of two regions of 6to 8 amino acids from the protein is determined for the production ofprimers for PCR amplification of a partial protease EOS DNA fragment.Alternatively, a longer degenerate oligonucleotide probe can besynthesized with a larger consecutive stretch of amino acid sequencedetermined. This oligonucleotide probe can be labeled and used to screena suitable cDNA or genomic library, under the appropriate stringency, toisolate DNA corresponding to protease EOS.

Once suitable amino acid sequences have been identified, the DNAsequences capable of encoding them are synthesized. Because the geneticcode is degenerate, more than one codon may be used to encode aparticular amino acid, and therefore, the amino acid sequence can beencoded by any of a set of similar DNA oligonucleotides. Only one memberof the set will be identical to the protease EOS sequence, but will becapable of hybridizing to protease EOS DNA even in the presence of DNAoligonucleotides with mismatches. The mismatched DNA oligonucleotidesmay still sufficiently hybridize to the protease EOS DNA to permitidentification and isolation of protease EOS encoding DNA. DNA isolatedby these methods can be used to screen DNA libraries from a variety ofcell types, from invertebrate and vertebrate sources, and to isolatehomologous genes.

Purified biologically active protease EOS may have several differentphysical forms. Protease EOS may exist as a full-length nascent orunprocessed polypeptide, or as partially processed polypeptides orcombinations of processed polypeptides. The full-length nascent proteaseEOS polypeptide may be posttranslationally modified by specificproteolytic cleavage events, which result in the formation of fragmentsof the full-length nascent polypeptide. A fragment, or physicalassociation of fragments may have the full biological activityassociated with protease EOS however, the degree of protease EOSactivity may vary between individual protease EOS fragments andphysically associated protease EOS polypeptide fragments.

The cloned protease EOS DNA obtained through the methods describedherein may be recombinantly expressed by molecular cloning into anexpression vector containing a suitable promoter and other appropriatetranscription regulatory elements, and transferred into prokaryotic oreukaryotic host cells to produce recombinant protease EOS protein.Techniques for such manipulations are fully described (Maniatis et al.(1989). 1-1626), and are well known in the art.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned copies of genes and the translation oftheir mRNAs in an appropriate host. Such vectors can be used to expresseukaryotic genes in a variety of hosts such as bacteria including E.coli, blue-green algae, plant cells, insect cells, fungal cellsincluding yeast cells, and animal cells.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells or bacteria-fungal cellsor bacteria-invertebrate cells. An appropriately constructed expressionvector should contain: an origin of replication for autonomousreplication in host cells, selectable markers, a limited number ofuseful restriction enzyme sites, a potential for high copy number, andactive promoters. A promoter is defined as a DNA sequence that directsRNA polymerase to bind to DNA and initiate RNA synthesis. A strongpromoter is one that causes mRNAs to be initiated at high frequency.Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.

A variety of mammalian expression vectors may be used to expressrecombinant protease EOS in mammalian cells. Commercially availablemammalian expression vectors which may be suitable for recombinantprotein expression, include but are not limited to, pCI Neo (Promega,Madison, Wis., Madison Wis.), pMAMneo (Clontech, Palo Alto, Calif.),pcDNA3 (InVitrogen, San Diego, Calif.), pMC1neo (Stratagene, La Jolla,Calif.), pXT1 (Stratagene, La Jolla, Calif.), pSG5 (Stratagene, LaJolla, Calif.), EBO-pSV2-neo (ATCC 37593) pBPV-1 (8-2) (ATCC 37110),pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC37565).

A variety of bacterial expression vectors may be used to expressrecombinant protease EOS in bacterial cells. Commercially availablebacterial expression vectors which may be suitable for recombinantprotein expression include, but are not limited to pET vectors (Novagen,Inc., Madison Wis.) and pQE vectors (Qiagen, Valencia, Calif.) pGEX(Pharmacia Biotech Inc., Piscataway, N.J.).

A variety of fungal cell expression vectors may be used to expressrecombinant protease EOS in fungal cells such as yeast. Commerciallyavailable fungal cell expression vectors which may be suitable forrecombinant protease EOS expression include but are not limited to pYES2(InVitrogen, San Diego, Calif.) and Pichia expression vector(InVitrogen, San Diego, Calif.).

A variety of insect cell expression systems may be used to expressrecombinant protease EOS in insect cells. Commercially availablebaculovirus transfer vectors which may be suitable for the generation ofa recombinant baculovirus for recombinant protein expression in Sf9cells include but are not limited to pFastBac1 (Life Technologies,Gaithersberg, Md.) pAcSG2 (Pharmingen, San Diego, Calif.) pBlueBacII(InVitrogen, San Diego, Calif.). In addition, a class of insect cellvectors that permit the expression of recombinant proteins in DrosophilaSchneider line 2 (S2) cells is also available (InVitrogen, San Diego,Calif.).

DNA encoding the protease EOS may be subcloned into an expression vectorfor expression in a recombinant host cell. Recombinant host cells may beprokaryotic or eukaryotic, including but not limited to bacteria such asE. coli, fungal cells such as yeast, mammalian cells including but notlimited to cell lines of human, bovine, porcine, monkey and rodentorigin, and insect cells including but not limited to Drosophila S2(ATCC CRL-1963) and silkworm Sf9 (ATCC CRL-1711), derived cell lines.Cell lines derived from mammalian species which may be suitable andwhich are commercially available, include but are not limited to, CV-1(ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCCCCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL171), L-cells, and HEK-293 (ATCC CRL 1573),

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, protoplast fusion, lipofection, and electroporation. Theexpression vector-containing cells are clonally propagated andindividually analyzed to determine whether they produce protease EOSprotein. Identification of protease ESO expressing host cell clones maybe done by several means, including but not limited to immunologicalreactivity with anti-protease EOS antibodies, and the presence of hostcell-associated EOS proteolytic activity.

Expression of protease EOS DNA may also be performed using in vitroproduced synthetic mRNA. Synthetic mRNA or mRNA isolated from proteaseEOS producing cells can be efficiently translated in various cell-freesystems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes,with microinjection into frog oocytes being generally preferred.

To determine the protease EOS DNA sequence(s) that yields optimal levelsof EOS proteolytic activity and/or EOS protein, protease EOS DNAmolecules including, but not limited to, the following can beconstructed: the full-length open reading frame of the protease EOS cDNAencoding the 30-kDa protein from approximately base 69 to approximatelybase 920 (these numbers correspond to first nucleotide of firstmethionine and last nucleotide before the first stop codon; FIG. 1) andseveral constructs containing portions of the cDNA encoding the EOSprotease. All constructs can be designed to contain none, all orportions of the 5′ or the 3′ untranslated region of the protease EOScDNA. Protease EOS activity and levels of protein expression can bedetermined following the introduction, both singly and in combination,of these constructs into appropriate host cells. Following determinationof the protease EOS DNA cassette yielding optimal expression intransient assays, this protease EOS DNA construct is transferred to avariety of expression vectors, for expression in host cells including,but not limited to, mammalian cells, baculovirus-infected insect cells,E. coli, and the yeast S. cerevisiae.

Host cell transfectants and microinjected oocytes may be used to assayboth the levels of protease EOS proteolytic activity and levels of EOSprotein by the following methods. In the case of recombinant host cells,this involves the co-transfection of one or possibly two or moreplasmids, containing the protease EOS DNA encoding one or more fragmentsor subunits. In the case of oocytes, this involves the co-injection ofsynthetic RNAs encoding protease EOS. Following an appropriate period oftime to allow for expression, cellular protein is metabolically labeledwith, for example ³⁵S-methionine for 24 hours, after which cell lysatesand cell culture supernatants are harvested and subjected toimmunoprecipitation with polyclonal antibodies directed against theprotease EOS protein.

Other methods for detecting protease EOS expression involve the directmeasurement of EOS proteolytic activity in whole cells transfected withprotease EOS CDNA or oocytes injected with protease EOS mRNA.Proteolytic activity can be measured by analyzing conditioned media orcell lysates by hydrolysis of a chromogenic or fluorogenic substrate. Inthe case of recombinant host cells expressing protease EOS, higherlevels of substrate hydrolysis would be observed relative to mocktransfected cells or cells transfected with expression vector lackingthe protease EOS DNA insert. In the case of oocytes, lysates orconditioned media from those injected with RNA encoding protease EOS,would show higher levels of substrate hydrolysis than those oocytesprogrammed with an irrelevant RNA.

Other methods for detecting proteolytic activity include, but are notlimited to, measuring the products of proteolytic degradation ofradiolabeled proteins (Coolican et al. (1986). J. Biol. Chem.261:4170-6), fluorometric (Lonergan et al. (1995). J. Food Sci. 60:72-3,78; Twining (1984). Anal. Biochem. 143:30-4) or colorimetric(Buroker-Kilgore and Wang (1993). Anal. Biochem. 208:387-92) analyses ofdegraded protein substrates. Zymography following SDS polyacrylamide gelelectrophoresis (Wadstroem and Smyth (1973). Sci. Tools 20:17-21), aswell as by fluorescent resonance energy transfer (FRET)-based methods(Ng and Auld (1989). Anal. Biochem. 183:50-6) are also methods used todetect proteolytic activity.

Levels of protease EOS protein in host cells can be quantitated byimmunoaffinity. protease EOS-specific affinity beads or proteaseEOS-specific antibodies are used to isolate for example ³⁵S-methioninelabeled or unlabelled protease EOS protein. Labeled protease EOS proteinis analyzed by SDS-PAGE. Unlabelled protease EOS protein is detected byWestern blotting, ELISA or RIA assays employing protease EOS specificantibodies.

Because the genetic code is degenerate, more than one codon may be usedto encode a particular amino acid, and therefore, the amino acidsequence can be encoded by any of a set of similar DNA oligonucleotides.Only one member of the set will be identical to the protease EOSsequence but will be capable of hybridizing to protease EOS DNA even inthe presence of DNA oligonucleotides with mismatches under appropriateconditions. Under alternate conditions, the mismatched DNAoligonucleotides may still hybridize to the protease EOS DNA to permitidentification and isolation of protease EOS encoding DNA.

DNA encoding protease EOS from a particular organism may be used toisolate and purify homologues of the protease EOS DNA from otherorganisms. To accomplish this, the first protease EOS DNA may be mixedwith a sample containing DNA encoding homologues of protease EOS underappropriate hybridization conditions. The hybridized DNA complex may beisolated and the DNA encoding the homologous DNA may be purifiedtherefrom.

It is known that there is a substantial amount of redundancy in thevarious codons that code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences that containalternative codons that code for the eventual translation of theidentical amino acid. For purposes of this specification, a sequencebearing one or more replaced codons will be defined as a degeneratevariation. Also included within the scope of this invention aremutations either in the DNA sequence or the translated protein which donot substantially alter the ultimate physical properties of theexpressed protein. For example, substitution of valine for leucine,arginine for lysine, or asparagine for glutamine may not cause a changein functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude, but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate or a receptor for a ligand.

Several recombinant serine protease purification procedures areavailable and suitable for use (Hansson et al. (1994). J. Biol. Chem.269:19420-6; Little et al. (1997). J. Biol. Chem. 272:25135-25142;Takayama et al. (1997). J. Biol. Chem. 272:21582-21588; Yamaoka et al.(1998). J. Biol. Chem. 273:11895-11901). As described above forpurification of protease EOS from natural sources, recombinant proteaseEOS may be purified from cell lysates and extracts, or from conditionedculture medium, by various combinations of, or individual application ofsalt fractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography. Following expression of proteaseEOS in a recombinant host cell, as is the case for many members of theS1 serine protease family, protease EOS protein may be recovered as aninactive zymogen precursor form which may require a limited proteolysisto become the proteolytically active.

A major drawback in the expression of full-length serine protease cDNAsfor biochemical and enzymological analyses is the overwhelming potentialfor the production of large amounts of the inactive zymogen. Thesezymogen precursors often have little or no significant proteolyticactivity and thus must be activated by either one of two methodscurrently available. One method relies on the autoactivation (Little etal. (1997). J. Biol. Chem. 272:25135-25142), which may occur inhomogeneous purified protease preparations under the correct set ofcircumstances. Investigators must rigorously evaluate these conditions,which often require high protein concentrations. The second method isthe use of a surrogate activating protease, such as trypsin, to cleavethe serine protease under investigation, and either inactivate (Takayamaet al. (1997). J. Biol. Chem. 272:21582-21588) or physically remove(Hansson et al. (1994). J. Biol. Chem. 269:19420-6) the contaminatingprotease following activation. In both methods however, the exactconditions must be established empirically and activating reactionsmonitored carefully, since inadequate activation or over-digestionleading to degradation and sample loss could always be possibleconsequences of these activating techniques. Investigators studyingparticular members of the S1 serine protease family have exploited theuse of restriction proteinases on the activation of expressed zymogensin bacteria (Wang et al. (1995). Biol. Chem. Hoppe-Seyler 376:681-4) andmammalian cells (Yamashiro et al. (1997). Biochim. Biophys. Acta1350:11-14). In one report, the authors successfully engineered thesecretion of proteolytically processed and activated murine granzyme Bby taking advantage of the endogenous yeast KEX2 signal peptidase in aPichia pastoris expression system (Pham et al. (1998). J. Biol. Chem.273:1629-1633). Another aspect of the present invention provides afusion gene comprising protease EOS that encodes a protease EOS thatfacilitates activation of the protease. DNA clones, including proteaseEOS DNA, are identified which encode proteins that, when expressed in arecombinant host, produce protein with the amino acid sequence ofprotease EOS, which may or may not possess a proteolytic activity. Theexpression of protease EOS DNA results in the reconstitution of theproperties observed in oocytes injected with protease EOS-encoding poly(A)⁺ RNA.

Recombinant protease EOS can be separated from other cellular proteinsby use of an immunoaffinity column made with monoclonal or polyclonalantibodies specific for full-length nascent protease EOS polypeptidefragments of protease EOS. Monospecific antibodies to protease EOS arepurified from mammalian antisera containing antibodies reactive againstprotease EOS or are prepared as monoclonal antibodies reactive withprotease EOS using the technique of (Kohler and Milstein (1976). Eur JImmunol 6:511-9). Monospecific antibody as used herein is defined as asingle antibody species or multiple antibody species with homogenousbinding characteristics for protease EOS. Homogenous binding as usedherein refers to the ability of the antibody species to bind to aspecific antigen or epitope, such as those associated with the proteaseEOS, as described above. Protease EOS specific antibodies are raised byimmunizing animals such as mice, rats, guinea pigs, rabbits, goats,horses and the like, with rabbits being preferred, with an appropriateconcentration of protease EOS either with or without an immune adjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.1 mg and about 1000 mg of protease EOSprotein or peptide(s), derived from the deduced protease EOS DNAsequence or perhaps by the chemical degradation or enzymatic digestionof the protease EOS protein itself, associated with an acceptable immuneadjuvant. Such acceptable adjuvants include, but are not limited to,Freund's complete, Freund's incomplete, alum-precipitate, water in oilemulsion containing Corynebacterium parvum and tRNA, or Titermax (CytRx,Norcross, Ga.). The initial immunization consists of protease EOSantigen in, preferably, Freund's complete adjuvant at multiple siteseither subcutaneously (SC), intraperitoneally (IP) or both. Each animalis bled at regular intervals, preferably weekly, to determine antibodytiter. The animals may or may not receive booster injections followingthe initial immunization. Those animals receiving booster injections aregenerally given an equal amount of the antigen in Freund's incompleteadjuvant by the same route. Booster injections are given at aboutthree-week intervals until maximal titers are obtained. At about 7 daysafter each booster immunization or about weekly after a singleimmunization, the animals are bled, the serum collected, and aliquotsare stored at about −20° C.

Monoclonal antibodies (MoAb) reactive with protease EOS are prepared byimmunizing inbred mice, preferably Balb/c, with protease EOS protein orpeptide(s), derived from the deduced protease EOS DNA sequence orperhaps by the chemical degradation or enzymatic digestion of theprotease EOS protein itself. The mice are immunized by the IP or SCroute with about 0.1 mg to about 10 mg, preferably about 1 mg, ofprotease EOS antigen in about 0.5 ml buffer or saline incorporated in anequal volume of an acceptable adjuvant, as discussed above. Freund'scomplete adjuvant is preferred. The mice receive an initial immunizationon day 0 and are rested for about 3 to about 30 weeks. Immunized miceare given one or more booster immunizations of about 0.1 to about 10 mgof protease EOS antigen in a buffer solution such as phosphate bufferedsaline by the intravenous (IV) route. Lymphocytes, from antibodypositive mice, preferably splenic lymphocytes, are obtained by removingspleens from immunized mice by standard procedures known in the art.Hybridoma cells are produced by mixing the splenic lymphocytes with anappropriate fusion partner, preferably myeloma cells, under conditionsthat will allow the formation of stable hybridomas. Fusion partners mayinclude, but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11;S-194 and Sp 2/0, with Sp 2/0 being generally preferred. The antibodyproducing cells and myeloma cells are fused in polyethylene glycol,about 1000 mol. wt., at concentrations from about 30% to about 50%.Fused hybridoma cells are selected by growth in hypoxanthine, thymidineand aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) byprocedures known in the art. Supernatant fluids are collected fromgrowth positive wells on about days 14, 18, and 21 and are screened forantibody production by an immunoassay such as solid phaseimmunoradioassay (SPIRA) using protease EOS or antigenic peptide(s) asthe antigen. The culture fluids are also tested in the Ouchterlonyprecipitation assay to determine the isotype of the MoAb. Hybridomacells from antibody positive wells are cloned by a technique such as thesoft agar technique of MacPherson, Soft Agar Techniques, in TissueCulture Methods and Applications, Kruse and Paterson, Eds., AcademicPress, 1973.

Monoclonal antibodies are produced in vivo by injection of pristaneprimed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10⁶ toabout 6×10⁶ hybridoma cells about 4 days after priming. Ascites fluid iscollected at approximately 8-12 days after cell transfer and themonoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-protease EOS MoAb is carried out by growingthe hybridoma in DMEM containing about 2% fetal calf serum to obtainsufficient quantities of the specific MoAb. The monoclonal antibodiesare purified by techniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of proteaseEOS in body fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for protease EOS polypeptide fragments,or full-length nascent protease EOS polypeptide. Specifically, it isreadily apparent to those skilled in the art that monospecificantibodies may be generated which are specific for only one or moreprotease EOS epitopes.

Protease EOS antibody affinity columns are made by adding the antibodiesto Affigel-10 (Bio-Rad), a gel support which is activated withN-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23 M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) and the cellculture supernatants or cell extracts containing protease EOS are slowlypassed through the column. The column is then washed with phosphatebuffered saline until the optical density (A₂₈₀) falls to background,then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). Thepurified protease EOS protein is then dialyzed against phosphatebuffered saline.

Protease EOS mRNA is expressed in retina, ovary, and stomach, where theencoded protease EOS protein may perform important functions duringnormal physiology, and possibly pathological states. Thus, modulators ofprotease EOS function could be used to treat disorders effecting thesetissues. We have also found that the protease EOS mRNA is expressedhuman platelets, and spleen which is a major site of blood cellmetabolism, as well as in leukocytes and more specifically ineosinophils. Eosinophilia, is a condition characterized by elevatedcirculating eosinophils, and is associated with numerous allergic statesincluding bronchial asthma (Gleich (1996). Allergol. Int. 45:35-44).Thus, protease EOS expression in platelets and certain cells of theimmune system suggests that it may play roles in hemostasis and a subsetof immune processes. Modulators of protease EOS function could thereforepotentially be used to treat disorders in hemostasis and/or to moderateparticular immune responses.

The present invention is also directed to methods for screening forcompounds that modulate the expression of DNA or RNA encoding protease Tas well as the function of protease T protein in vivo. Compounds thatmodulate these activities may be DNA, RNA, peptides, proteins, ornon-proteinaceous organic molecules. Compounds may modulate byincreasing or attenuating the expression of DNA or RNA encoding proteaseT, or the function of protease T protein. Compounds that modulate theexpression of DNA or RNA encoding protease T or the function of proteaseT protein may be detected by a variety of assays. The assay may be asimple “yes/no” assay to determine whether there is a change inexpression or function. The assay may be made quantitative by comparingthe expression or function of a test sample with the levels ofexpression or function in a standard sample. Modulators identified inthis process are potentially useful as therapeutic agents. Methods fordetecting compounds that modulate protease T proteolytic activitycomprise combining compound, protease T and a suitable labeled substrateand monitoring an effect of the compound on the protease by changes inthe around of substrate as a function of time. Labeled substratesinclude, but are not limited to, substrate that are radiolabeled(Coolican et al. (1986). J. Biol. Chem. 261:4170-6), fluorometric(Lonergan et al. (1995). J. Food Sci. 60:72-3, 78; Twining (1984). Anal.Biochem. 143:30-4) or calorimetric (Buroker-Kilgore and Wang (1993).Anal. Biochem. 208:387-92). Zymography following SDS polyacrylamide gelelectrophoresis (Wadstroem and Smyth (1973). Sci. Tools 20:17-21), aswell as by fluorescent resonance energy transfer (FRET)-based methods(Ng and Auld (1989). Anal. Biochem. 183:50-6) are also methods used todetect compounds that modulate protease T proteolytic activity.Compounds that are agonists will increase the rate of substratedegradation and will result in less remaining substrate as a function oftime. Compounds that are antagonists will decrease the rate of substratedegradation and will result in greater remaining substrate as a functionof time.

Kits containing protease EOS DNA or RNA, antibodies to protease EOS, orprotease EOS protein may be prepared. Such kits are used to detect DNAthat hybridizes to protease EOS DNA or to detect the presence ofprotease EOS protein or peptide fragments in a sample. Suchcharacterization is useful for a variety of purposes including but notlimited to forensic analyses, diagnostic applications, andepidemiological studies.

The present invention is also directed to methods for screening forcompounds that modulate the expression of DNA or RNA encoding proteaseEOS as well as the function of protease EOS protein in vivo. Compoundsthat modulate these activities may be DNA, RNA, peptides, proteins, ornon-proteinaceous organic molecules. Compounds may modulate byincreasing or attenuating the expression of DNA or RNA encoding proteaseEOS, or the function of protease EOS protein. Compounds that modulatethe expression of DNA or RNA encoding protease EOS or the function ofprotease EOS protein may be detected by a variety of assays. The assaymay be a simple “yes/no” assay to determine whether there is a change inexpression or function. The assay may be made quantitative by comparingthe expression or function of a test sample with the levels ofexpression or function in a standard sample. Modulators identified inthis process are potentially useful as therapeutic agents. Methods fordetecting compounds that modulate protease EOS proteolytic activitycomprise combining compound, protease EOS and a suitable labeledsubstrate and monitoring an effect of the compound on the protease bychanges in the amount of substrate as a function of time. Labeledsubstrates include, but are not limited to, substrates that areradiolabeled (Coolican et al. (1986). J. Biol. Chem. 261:4170-6),fluorometric (Lonergan et al. (1995). J. Food Sci. 60:72-3, 78; Twining(1984). Anal. Biochem. 143:30-4) or colorimetric (Buroker-Kilgore andWang (1993). Anal. Biochem. 208:387-92). Zymography following SDSpolyacrylamide gel electrophoresis (Wadstroem and Smyth (1973). Sci.Tools 20:17-21), as well as by fluorescent resonance energy transfer(FRET)-based methods (Ng and Auld (1989). Anal. Biochem. 183:50-6) arealso methods used to detect compounds that modulate protease EOSproteolytic activity. Compounds that are agonists will increase the rateof substrate degradation and will result in less remaining substrate asa function of time. Compounds that are antagonists will decrease therate of substrate degradation and will result in greater remainingsubstrate as a function of time.

Nucleotide sequences that are complementary to the protease EOS encodingDNA sequence can be synthesized for antisense therapy. These antisensemolecules may be DNA, stable derivatives of DNA such asphosphorothioates or methylphosphonates, RNA, stable derivatives of RNAsuch as 2′-O-alkylRNA, or other protease EOS antisense oligonucleotidemimetics. protease EOS antisense molecules may be introduced into cellsby microinjection, liposome encapsulation or by expression from vectorsharboring the antisense sequence. Protease EOS antisense therapy may beparticularly useful for the treatment of diseases where it is beneficialto reduce protease EOS expression or activity.

Protease EOS gene therapy may be used to introduce protease EOS into thecells of target organisms. The protease EOS gene can be ligated intoviral vectors that mediate transfer of the protease EOS DNA by infectionof recipient host cells. Suitable viral vectors include retrovirus,adenovirus, adeno-associated virus, herpes virus, vaccinia virus,poliovirus and the like. Alternatively, protease EOS DNA can betransferred into cells for gene therapy by non-viral techniquesincluding receptor-mediated targeted DNA transfer using ligand-DNAconjugates or adenovirus-ligand-DNA conjugates, lipofection membranefusion or direct microinjection. These procedures and variations thereofare suitable for ex vivo as well as in vivo protease EOS gene therapy.Protease EOS gene therapy may be particularly useful for the treatmentof diseases where it is beneficial to elevate protease EOS expression oractivity.

Pharmaceutically useful compositions comprising protease EOS DNA,protease EOS RNA, or protease EOS protein, or modulators of protease EOSactivity, may be formulated according to known methods such as by theadmixture of a pharmaceutically acceptable carrier. Examples of suchcarriers and methods of formulation may be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the protein, DNA, RNA, or modulator.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose disorders inwhich modulation of protease EOS-related activity is indicated. Theeffective amount may vary according to a variety of factors such as theindividual's condition, weight, sex and age. Other factors include themode of administration. The pharmaceutical compositions may be providedto the individual by a variety of routes such as subcutaneous, topical,oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties that are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages defined by routine testing in order toobtain optimal inhibition of the protease EOS activity while minimizingany potential toxicity. In addition, co-administration or sequentialadministration of other agents may be desirable.

The protease EOS may be formulated as an active ingredient innon-pharmaceutical commercial products including laundry detergents,skin care lotions or creams. In these formulations the protease EOS isutilized to degrade proteins to increase the efficacy of the product.For example, in laundry detergent formulations inclusion of the proteaseEOS would act as a “stain remover” by degrading proteacious contaminantsfrom fabric such that the organic compound would become more soluble indetergent and water. Protease EOS can be included in skin care productsto aid in desquamation, the process of elimination of the superficiallayers of the stratum corneum. An additional benefit of utilizing theprotease EOS in non-pharmaceutical commercial formulations is that it isnot likely to induce allergic response in sensitive individuals sincethe protease EOS is of human origin.

The present invention also has the objective of providing suitabletopical, oral, systemic and parenteral pharmaceutical formulations foruse in the novel methods of treatment of the present invention. Thecompositions containing compounds or modulators identified according tothis invention as the active ingredient for use in the modulation ofprotease EOS activity can be administered in a wide variety oftherapeutic dosage forms in conventional vehicles for administration.For example, the compounds or modulators can be administered in suchoral dosage forms as tablets, capsules (each including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, solutions, suspensions, syrups and emulsions, or byinjection. Likewise, they may also be administered in intravenous (bothbolus and infusion), intraperitoneal, subcutaneous, topical with orwithout occlusion, or intramuscular form, all using forms well known tothose of ordinary skill in the pharmaceutical arts. An effective butnon-toxic amount of the compound desired can be employed as a proteaseEOS modulating agent.

The daily dosage of the products may be varied over a wide range from0.01 to 1,000 mg per patient, per day. For oral administration, thecompositions are preferably provided in the form of scored or unscoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 100 mg/kg of body weight per day. The rangeis more particularly from about 0.001 mg/kg to 10 mg/kg of body weightper day. The dosages of the protease EOS modulators are adjusted whencombined to achieve desired effects. On the other hand, dosages of thesevarious agents may be independently optimized and combined to achieve asynergistic result wherein the pathology is reduced more than it wouldbe if either agent were used alone.

Advantageously, compounds or modulators of the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.Furthermore, compounds or modulators for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds or modulators of the presentinvention is selected in accordance with a variety of factors includingtype, species, age, weight, sex and medical condition of the patient;the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

In the methods of the present invention, the compounds or modulatorsherein described in detail can form the active ingredient, and aretypically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents that may be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations, which generally containsuitable preservatives, are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as, eg.,alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils,mineral oil, PPG2 myristyl propionate, and the like, to form, eg.,alcoholic solutions, topical cleansers, cleansing creams, skin gels,skin lotions, and shampoos in cream or gel formulations.

The compounds or modulators of the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds or modulators of the presentinvention may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinyl-pyrrolidone, pyrancopolymer, polyhydroxypropylmethacryl-amidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolysinesubstituted with palmitoyl residues. Furthermore, the compounds ormodulators of the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels.

For oral administration, the compounds or modulators may be administeredin capsule, tablet, or bolus form or alternatively they can be mixed inthe animals feed. The capsules, tablets, and boluses are comprised ofthe active ingredient in combination with an appropriate carrier vehiclesuch as starch, talc, magnesium stearate, or di-calcium phosphate. Theseunit dosage forms are prepared by intimately mixing the activeingredient with suitable finely-powdered inert ingredients includingdiluents, fillers, disintegrating agents, and/or binders such that auniform mixture is obtained. An inert ingredient is one that will notreact with the compounds or modulators and which is non-toxic to theanimal being treated. Suitable inert ingredients include starch,lactose, talc, magnesium stearate, vegetable gums and oils, and thelike. These formulations may contain a widely variable amount of theactive and inactive ingredients depending on numerous factors such asthe size and type of the animal species to be treated and the type andseverity of the infection. The active ingredient may also beadministered as an additive to the feed by simply mixing the compoundwith the feedstuff or by applying the compound to the surface of thefeed. Alternatively the active ingredient may be mixed with an inertcarrier and the resulting composition may then either be mixed with thefeed or fed directly to the animal. Suitable inert carriers include cornmeal, citrus meal, fermentation residues, soya grits, dried grains andthe like. The active ingredients are intimately mixed with these inertcarriers by grinding, stirring, milling, or tumbling such that the finalcomposition contains from 0.001 to 5% by weight of the activeingredient.

The compounds or modulators may alternatively be administeredparenterally via injection of a formulation consisting of the activeingredient dissolved in an inert liquid carrier. Injection may be eitherintramuscular, intraruminal, intratracheal, or subcutaneous. Theinjectable formulation consists of the active ingredient mixed with anappropriate inert liquid carrier. Acceptable liquid carriers include thevegetable oils such as peanut oil, cottonseed oil, sesame oil and thelike as well as organic solvents such as solketal, glycerol formal andthe like. As an alternative, aqueous parenteral formulations may also beused. The vegetable oils are the preferred liquid carriers. Theformulations are prepared by dissolving or suspending the activeingredient in the liquid carrier such that the final formulationcontains from 0.005 to 10% by weight of the active ingredient.

Topical application of the compounds or modulators is possible throughthe use of a liquid drench or a shampoo containing the instant compoundsor modulators as an aqueous solution or suspension. These formulationsgenerally contain a suspending agent such as bentonite and normally willalso contain an antifoaming agent. Formulations containing from 0.005 to10% by weight of the active ingredient are acceptable. Preferredformulations are those containing from 0.01 to 5% by weight of theinstant compounds or modulators. Proteases are used in non-naturalenvironments for various commercial purposes including laundrydetergents, food processing, fabric processing, and skin care products.In laundry detergents, the protease is employed to break down organic,poorly soluble compounds to more soluble forms that can be more easilydissolved in detergent and water. In this capacity the protease acts asa “stain remover.” Examples of food processing include tenderizing meatsand producing cheese. Proteases are used in fabric processing, forexample, to treat wool in order prevent fabric shrinkage. Proteases maybe included in skin care products to remove scales on the skin surfacethat build up due to an imbalance in the rate of desquamation. Commonproteases used in some of these applications are derived fromprokaryotic or eukaryotic cells that are easily grown for industrialmanufacture of their enzymes, for example a common species used isBacillus as described in U.S. Pat. No. 5,217,878. Alternatively, U.S.Pat. No. 5,278,062 describes serine proteases isolated from a fungus,Tritirachium album, for use in laundry detergent compositions.Unfortunately use of some proteases is limited by their potential tocause allergic reactions in sensitive individuals or by reducedefficiency when used in a non-natural environment. It is anticipatedthat protease proteins derived from non-human sources would be morelikely to induce an immune response in a sensitive individual. Becauseof these limitations, there is a need for alternative proteases that areless immunogenic to sensitive individuals and/or provides efficientproteolytic activity in a non-natural environment. The advent ofrecombinant technology allows expression of any species' proteins in ahost suitable for industrial manufacture.

Another aspect of the present invention relates to compositionscomprising the protease EOS and an acceptable carrier. The compositionmay be any variety of compositions that requires a protease component.Particularly preferred are compositions that may come in contact withhumans, for example, through use or manufacture. The use of the proteaseEOS of the present invention is believed to reduce or eliminate theimmunogenic response users and/or handlers might otherwise experiencewith a similar composition containing a known protease, particularly aprotease of non-human origin. Preferred compositions are skin carecompositions and laundry detergent compositions.

Herein, “acceptable carriers” includes, but is not limited to,cosmetically-acceptable carriers, pharmaceutically-acceptable carriers,and carriers acceptable for use in cleaning compositions.

Skin Care Compositions

Skin care compositions of the present invention preferably comprise, inaddition to the protease EOS, a cosmetically- orpharmaceutically-acceptable carrier.

Herein, “cosmetically-acceptable carrier” means one or more compatiblesolid or liquid filler diluents or encapsulating substances which aresuitable for use in contact with the skin of humans and lower animalswithout undue toxicity, incompatibility, instability, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio.

Herein, “pharmaceutically-acceptable” means one or more compatibledrugs, medicaments or inert ingredients which are suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, incompatibility, instability, irritation, allergic response,and the like, commensurate with a reasonable benefit/risk ratio.Pharmaceutically-acceptable carriers must, of course, be of sufficientlyhigh purity and sufficiently low toxicity to render them suitable foradministration to the mammal being treated.

Herein, “compatible” means that the components of the cosmetic orpharmaceutical compositions are capable of being commingled with theprotease EOS, and with each other, in a manner such that there is nointeraction which would substantially reduce the cosmetic orpharmaceutical efficacy of the composition under ordinary usesituations.

Preferably the skin care compositions of the present invention aretopical compositions, i.e., they are applied topically by the directlaying on or spreading of the composition on skin. Preferably suchtopical compositions comprise a cosmetically- orpharmaceutically-acceptable topical carrier.

The topical composition may be made into a wide variety of producttypes. These include, but are not limited to, lotions, creams, beachoils, gels, sticks, sprays, ointments, pastes, mousses, and cosmetics;hair care compositions such as shampoos and conditioners (for, e.g.,treating/preventing dandruff); and personal cleansing compositions.These product types may comprise several carrier systems including, butnot limited to, solutions, emulsions, gels and solids.

Preferably the carrier is a cosmetically- or pharmaceutically-acceptableaqueous or organic solvent. Water is a preferred solvent. Examples ofsuitable organic solvents include: propylene glycol, polyethylene glycol(200-600), polypropylene glycol (425-2025), propylene glycol-14 butylether, glycerol, 1,2,4butanetriol, sorbitol esters, 1,2,6-hexanetriol,ethanol, isopropanol, butanediol, and mixtures thereof. Such solutionsuseful in the present invention preferably contain from about 0.001% toabout 25% of the protease EOS, more preferably from about 0.1% to about10% more preferably from about 0.5% to about 5%; and preferably fromabout 50% to about 99.99% of an acceptable aqueous or organic solvent,more preferably from about 90% to about 99%.

Skin care compositions of the present invention may further include awide variety of additional oil-soluble materials and/or water-solublematerials conventionally used in topical compositions, at theirart-established levels. Such additional components include, but are notlimited to: thickeners, pigments, fragrances, humectants, proteins andpolypeptides, preservatives, pacifiers, penetration enhancing agents,collagen, hylauronic acid, elastin, hydrolysates, primrose oil, jojobaoil, epidermal growth factor, soybean saponins, mucopolysaccharides,Vitamin A and derivatives thereof, Vitamin B2, biotin, pantothenic acid,Vitamin D, and mixtures thereof.

Cleaning Compositions

Cleaning compositions of the present invention preferably comprise, inaddition to the protease EOS, a surfactant. The cleaning composition maybe in a wide variety of forms, including, but not limited to, hardsurface cleaning compositions, dishcare cleaning compositions, andlaundry detergent compositions.

Preferred cleaning compositions are laundry detergent compositions. Suchlaundry detergent compositions include, but not limited to, granular,liquid and bar compositions. Preferably, the laundry detergentcomposition further comprises a builder.

The laundry detergent composition of the present invention contains theprotease EOS at a level sufficient to provide a “cleaning-effectiveamount”. The term “cleaning effective amount” refers to any amountcapable of producing a cleaning, stain removal, soil removal, whitening,deodorizing, or freshness improving effect on substrates such asfabrics, dishware and the like. In practical terms for currentcommercial preparations, typical amounts are up to about 5 mg by weight,more typically 0.01 mg to 3 mg, of active enzyme per gram of thedetergent composition. Stated another way, the laundry detergentcompositions herein will typically comprise from 0.001% to 5%,preferably 0.01%-3%, more preferably 0.01% to 1% by weight of rawprotease EOS preparation. Herein, “raw protease EOS preparation” refersto preparations or compositions in which the protease EOS is containedin prior to its addition to the laundry detergent composition.Preferably, the protease EOS is present in such raw protease EOSpreparations at levels sufficient to provide from 0.005 to 0.1 Ansonunits (AU) of activity per gram of raw protease EOS preparation. Forcertain detergents, such as in automatic dishwashing, it maybe desirableto increase the active protease EOS content of the raw protease EOSpreparation in order to minimize the total amount of non-catalyticallyactive materials and thereby improve spotting/filming or otherend-results. Higher active levels may also be desirable in highlyconcentrated detergent formulations.

Preferably, the laundry detergent compositions of the present invention,including but not limited to liquid compositions, may comprise fromabout 0.001% to about 10%, preferably from about 0.005% to about 8%,most preferably from about 0.01% to about 6%, by weight of an enzymestabilizing system. The enzyme stabilizing system can be any stabilizingsystem that is compatible with the protease EOS, or any other additionaldetersive enzymes that may be included in the composition. Such a systemmay be inherently provided by other formulation actives, or be addedseparately, e.g., by the formulator or by a manufacturer ofdetergent-ready enzymes. Such stabilizing systems can, for example,comprise calcium ion, boric acid, propylene glycol, short chaincarboxylic acids, boronic acids, and mixtures thereof, and are designedto address different stabilization problems depending on the type andphysical form of the detergent composition.

The detergent composition also comprises a detersive surfactant.Preferably the detergent composition comprises at least about 0.01% of adetersive surfactant; more preferably at least about 0.1%; morepreferably at least about 1%; more preferably still, from about 1% toabout 55%.

Preferred detersive surfactants are cationic, anionic, nonionic,ampholytic, zwifterionic, and mixtures thereof, further described hereinbelow. Nonlimiting examples of detersive surfactants useful in thedetergent composition include, the conventional C11-C18 alkyl benzenesulfonates (“LAS”) and primary, branched-chain and random C10-C20 alkylsulfates (“AS”), the C10-C18 secondary (2,3) alkyl sulfates of theformula CH₃(CH₂)x(CHOSO₃—M+) CH₃ and CH₃ (CH₂)_(y)(CHOSO₃—M+) CH₂CH₃where x and (y+1) are integers of at least about 7, preferably at leastabout 9, and M is a water-solubilizing cation, especially sodium,unsaturated sulfates such as oleyl sulfate, the C10-C18 alkyl alkoxysulfates (“AExS”; especially EO 1-7 ethoxy sulfates), C10-C18 alkylalkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), theC10-C18 glycerol ethers, the C10-C18 alkyl polyglycosides and theircorresponding sulfated polyglycosides, and C12-C18 alpha-sulfonatedfatty acid esters. If desired, the conventional nonionic and amphotericsurfactants such as the C12-C18 alkyl ethoxylates (“AE”) including theso-called narrow peaked alkyl Ethoxylates and C6-C12 alkyl phenolalkoxylates (especially ethoxylates and mixed ethoxy/propoxy), C12-C18betaines and solfobetaines (“sultaines”), C10-C18 amine oxides, and thelike, can also be included in the overall compositions. The C10-C18N-alkyl polyhydroxy fatty acid amides can also be used. Typical examplesinclude the C12-C18 N-methylglucamides. See WO 9,206,154.

Other sugar-derived surfactants include the N-alkoxy polyhydroxy fattyacid amides, such as C10-C18 N-(3-methoxypropyl) glucamide. The N-propylthrough N-hexyl C12-C18 glucamides can be used for low sudsing. C10-C20conventional soaps may also be used. If high sudsing is desired, thebranched-chain C10-C16 soaps may be used. Mixtures of anionic andnonionic surfactants are especially useful. Other conventional usefulsurfactants are listed in standard texts. Detergent builders are alsoincluded in the laundry detergent composition to assist in controllingmineral hardness. Inorganic as well as organic builders can be used.Builders are typically used in fabric laundering compositions to assistin the removal of particulate soils.

The level of builder can vary widely depending upon the end use of thecomposition and its desired physical form. When present, thecompositions will typically comprise at least about 1% builder. Liquidformulations typically comprise from about 5% to about 50%, moretypically about 5% to about 30%, by weight, of detergent builder.Granular formulations typically comprise from about 10% to about 80%,more typically from about 15% to about 50% by weight, of the detergentbuilder. Lower or higher levels of builder, however, are not meant to beexcluded.

Inorganic or P-containing detergent builders include, but are notlimited to, the alkali metal, ammonium and alkanolammonium salts ofpolyphosphates (exemplified by the tripolyphosphates, pyrophosphates,and glassy polymeric meta-phosphates), phosphonates, phytic acid,silicates, carbonates (including bicarbonates and sesquicarbonates),sulphates, and aluminosilicates. However, non-phosphate builders arerequired in some locales. Importantly, the compositions herein functionsurprisingly well even in the presence of the so-called “weak” builders(as compared with phosphates) such as citrate, or in the so-called“underbuilt” situation that may occur with zeolite or layered silicatebuilders.

Examples of silicate builders are the alkali metal silicates,particularly those having a SiO2:Na2O ration in the range 1.6:1 to 3.2:1and layered silicates, such as the layered sodium silicates described inU.S. Pat. No. 4,664,839, issued May 12, 1987 to H. P. Rieck. NaSKS-6 isthe trademark for a crystalline layered silicate marketed by Hoechst(commonly abbreviated herein as “SKS-6”). Unlike zeolite builders, theNa SKS-6 silicate builder does not contain aluminum. NaSKS-6 has thedelta-Na2SiO5 morphology form of layered silicate. It can be prepared bymethods such as those described in German DE-A-3,417,649 andDE-A-3,742,043. SKS-6 is a highly preferred layered silicate for useherein, but other such layered silicates, such as those having thegeneral formula NaMSixO2x+1 yH20 wherein M is sodium or hydrogen, x is anumber from 1.9 to 4, preferably 2, and y is a number from 0 to 20,preferably 0 can be used herein. Various other layered silicates fromHoechst include NaSKS-5, NaSKS-7 and NaSKS-1 1, as the alpha, beta andgamma forms. As noted above, the delta-Na2SiO5 (NaSKS-6 form) is mostpreferred for use herein. Other silicates may also be useful such as forexample magnesium silicate, which can serve as a crispening agent ingranular formulations, as a stabilizing agent for oxygen bleaches, andas a component of suds control systems. Examples of carbonate buildersare the alkaline earth and alkali metal carbonates as disclosed inGerman Patent Application No. 2,321,001 published on Nov. 15, 1973.

Aluminosilicate builders are useful in the present invention.Aluminosilicate builders are of great importance in most currentlymarketed heavy duty granular detergent compositions, and can also be asignificant builder ingredient in liquid detergent formulations.Aluminosilicate builders include those having the empirical formula:

M_(z)(zAlO₂)_(y)—xH₂O

wherein z and y are integers of at least 6, the molar ratio of z to y isin the range from 1.0 to about 0.5, and x is an integer from about 15 toabout 264.

Useful aluminosilicate ion exchange materials are commerciallyavailable. These aluminosilicates can be crystalline or amorphous instructure and can be naturally-occurring aluminosilicates orsynthetically derived. A method for producing aluminosilicate ionexchange materials is disclosed in U.S. Pat. No. 3,985,669, Krummel, etal, issued Oct. 12, 1976. Preferred synthetic crystallinealuminosilicate ion exchange materials useful herein are available underthe designations Zeolite A, Zeolite P (b), Zeolite MAP and Zeolite X. Inan especially preferred embodiment, the crystalline aluminosilicate ionexchange material has the formula:

Na₁₂[(AlO₂)₁₂(SiO₂)₁₂].xH₂O

wherein x is from about 20 to about 30, especially about 27. Thismaterial is known as Zeolite A. Dehydrated zeolites (x=0−10) may also beused herein. Preferably, the aluminosilicate has a particle size ofabout 0.1-10 microns in diameter.

Organic detergent builders suitable for the purposes of the presentinvention include, but are not restricted to, a wide variety ofpolycarboxylate compounds. As used herein, “polycarboxylate” refers tocompounds having a plurality of carboxylate groups, preferably at least3 carboxylates. Polycarboxylate builder can generally be added to thecomposition in acid form, but can also be added in the form of aneutralized salt. When utilized in salt form, alkali metals, such assodium, potassium, and lithium, or alkanolammonium salts are preferred.

Included among the polycarboxylate builders are a variety of categoriesof useful materials. One important category of polycarbonate buildersencompasses the ether polycarboxylates, including oxydisuccinate, asdisclosed in Berg, U.S. Pat. No. 3,128,287, issued Apr. 7, 1964, andLamberti et al., U.S. Pat. No. 3,635,830, issued Jan. 18, 1972. See also“TMSFTDS” builders of U.S. Pat. No. 4,663,071, issued to Bush et al., onMay 5, 1987. Suitable ether polycarboxylates also include cycliccompounds, particularly alicyclic compounds, such as those described inU.S. Pat. No. 3,923,679 to Rapko, issued Dec. 2, 1975; U.S. Pat. No.3,835,163 to Rapko, issued Sep. 10, 1974; U.S. Pat. No. 4,158,635 toCrutchfield et al., issued Jun. 19, 1979; U.S. Pat. No. 4,120,874 toCrutchfield et al., issued Oct. 17, 1978; and U.S. Pat. No. 4,102,903 toCrutchfield et al., issued Jul. 25, 1978.

Other useful detergency builders include the etherhydroxypolycarboxylates, copolymers of maleic anhydride with ethylene orvinyl methyl ether, 1,3,5-trihydroxy benzene-2,4,6-t6sulphonic acid, andcarboxymethyloxysuccinic acid, the various alkali metal, ammonium andsubstituted ammonium salts of polyacetic acids such as. ethylenediaminetetraacetic acid and nitrilotriacetic acid, as well as polycarboxylatessuch as Mellitic acid, succinic acid, oxydisuccinic acid, polymaleicacid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid,and soluble salts thereof, citrate builders, e.g., citric acid andsoluble salts thereof (particularly sodium salt), are polycarboxylatebuilders of particular importance for heavy-duty liquid detergentformulations due to their availability from renewable resources andtheir biodegradability. Citrates can also be used in granularcompositions, especially in combination with zeolite and/or layeredsilicate builders. Oxydisuccinates are also especially useful in suchcompositions and combinations.

Also suitable in the detergent compositions of the present invention arethe 3,3-dicarboxy-4-oxa-1,6-hexanedioates and the related compoundsdisclosed in U.S. Pat. No. 4,566,984 to Bush, issued Jan. 28, 1986.Useful succinic acid builders include the C5-C20 alkyl and alkenylsuccinic acids and salts thereof. A particularly preferred compound ofthis type is dodecenylsuccinic acid. Specific examples of succinatebuilders include: laurylsuccinate, myristylsuccinate, paimitylsuccinate,2-dodecenylsuccinate (preferred), 2pentadecenylsuccinate, and the like.Lauryisuccinates are the preferred builders of this group, and aredescribed in European Patent Application 200,263 to Barrat et al.,published Nov. 5, 1986.

Other suitable polycarboxylates are disclosed in U.S. Pat. No.4,144,226, Crutchfield et al, issued Mar. 13, 1979 and in U.S. Pat. No.3,308,067, Diehl, issued Mar. 7, 1967. See also U.S. Pat. No. 3,723,322to Diehl, issued Mar. 27, 1973.

Fatty acids, e.g., C12-C18 monocarboxylic acids, can also beincorporated into the compositions alone, or in combination with theaforesaid builders, especially citrate and/or the succinate builders, toprovide additional builder activity. Such use of fatty acids willgenerally result in a diminution of sudsing, which should be taken intoaccount by the formulator.

In situations where phosphorus-based builders can be used, andespecially in the formulation of bars used for hand-launderingoperations, the various alkali metal phosphates such as the well-knownsodium tripolyphosphates, sodium pyrophosphate and sodium orthophosphatecan be used. Phosphonate builders such asethane-1-hydroxy-1,1-diphosphate and other known phosphonates (see, forexample, U.S. Pat. No. 3,159,581 to Diehl, issued Dec. 1, 1964; U.S.Pat. No. 3,213,030 to Diehl, issued Oct. 19, 1965; U.S. Pat. No.3,400,148 to Quimby, issued Sep. 3, 1968; U.S. Pat. No. 3,422,021 toRoy, issued Jan. 14, 1969; and U.S. Pat. No. 3,422,137 to Quimby, issuedJan. 4, 1969) can also be used. Additional components which may be usedin the laundry detergent compositions of the present invention include,but are not limited to: alkoxylated polycarboxylates (to provide, e.g.,additional grease stain removal performance), bleaching agents, bleachactivators, bleach catalysts, brighteners, chelating agents, clay soilremoval/anti-redeposition agents, dye transfer inhibiting agents,additional enzymes (including lipases, amylases, hydrolases, and otherproteases), fabric softeners, polymeric soil release agents, polymericdispersing agents, and suds suppressors.

The compositions herein may further include one or more other detergentadjunct materials or other materials for assisting or enhancing cleaningperformance, treatment of the substrate to be cleaned, or to modify theaesthetics of the detergent composition (e.g., perfumes, colorants,dyes, etc.). The detergent compositions herein may further compriseother known detergent cleaning components including alkoxylatedpolycarboxylates, bleaching compounds, brighteners, chelating agents,clay soil removal/antiredeposition agents, dye transfer inhibitingagents, enzymes, enzyme stabilizing systems, fabric softeners, polymericsoil release agents, polymeric dispersing agents, suds suppressors. Thedetergent composition may also comprise other ingredients includingcarriers, hydrotropes, processing aids, dyes or pigments, solvents forliquid formulations, solid fillers for bar compositions.

Method of Treating or Preventing Skin Flaking

Another aspect of the present invention relates to a method of treatingor preventing skin flaking. The method comprises topical application ofa safe and effective amount of a composition comprising the proteaseEOS.

Herein, “safe and effective amount” means an amount of protease EOS highenough to provide a significant positive modification of the conditionto be treated, but low enough to avoid serious side effects (at areasonable benefit/risk ratio), within the scope of sound medicaljudgment. A safe and effective amount of protease EOS will vary with theparticular condition being treated, the age and physical condition ofthe subject being treated, the severity of the condition, the durationof the treatment, the nature of concurrent therapy and like factors.

Suitable compositions for use in the subject method include theabove-described skin care compositions, including hair care compositions(for example, treating/preventing dandruff caused by skin flaking.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1

Plasmid Manipulations

All molecular biological methods were in accordance with thosepreviously described (Maniatis et al. (1989). 1-1626). Oligonucleotideswere purchased from Ransom Hill Biosciences (Ransom Hill, Calif.) andall restriction endonucleases and other DNA modifying enzymes were fromNew England Biolabs (Beverly, Mass.) unless otherwise specified. Theprotease EOS expression construct was made in the baculovirus expressionvector pFastBac1 (Life Technologies, Gaithersberg, Md.) as describedbelow. All construct manipulations were confirmed by dye terminatorcycle sequencing using Allied Biosystems 377 fluorescent sequencers(Perkin Elmer, Foster City, Calif.).

Acquisition of Protease EOS cDNA

Eosinophil RNA was isolated from pooled diseased eosinophils obtainedfrom allergic asthmatic individuals. Eosinophils were lysed immediatelyfollowing collection and purification in a buffer containing guanidiniumisothiocyanate. The lysate was spun through CsCl to obtain total RNA,followed by poly A isolation using Oligotex latex beads. The cDNAsynthesis was initiated using a NotI-oligo(dT) primer anddouble-stranded cDNA was blunted, ligated to Sal I adaptors, digestedwith Not I, size-selected, and cloned into the Not I and Sal I sites ofthe pSPORT1 vector (Life Technologies, Gaithersberg, Md.). A clone,corresponding to the full-length protease EOS CDNA, contained an openreading frame of 855 nucleotides (FIG. 1), and had homology to other S1serine proteases. This clone is also likely to contain the entire 3′untranslated since an AATAAA motif resides 18 nucleotides upstream of apoly A stretch of 50 nucleotides (in this particular clone, data notshown) a subset of which is presented in FIG. 1. Homology searches ofthe Genbank database with the protease EOS cDNA indicated that this wasa novel cDNA but had identity with the human cosmid clone (407D8,Genbank accession #AC005570), which maps to chromosome 16p13.3,indicating the position of protease EOS gene. The deduced open readingframe encodes a preproEOS protein of 284 amino acids (FIG. 1), with anestimated molecular mass (M_(r)) of about 30-Kd, and a strong homologyto other serine proteases. The catalytic triad residues H, D and S arelocated at positions 77, 126 and 231, respectively. The zymogenactivation sequence MSSR-IVGG (positions 33 to 40) is very similar tothat of other S1 serine proteases and predicts a mature protein of 284amino acids. A signal peptide of 22 amino acids is predicted bystatistical method (Von Heijne (1986). Nucleic Acids Res. 14:4683-90)indicating a pro peptide of 14 amino acids. FASTA homology against theSWISS-PROT database indicates that the top match to protease EOS is thehuman prostasin precursor EC 3.4.21 SW:Q16651 (Yu et al. (1995). J.Biol. Chem. 270:13483-9; Yu et al. (1996). Genomics 32:334-40) (47.1%identity in 276 aa. overlap). The next match is dog tryptase precursorEC 3.4.21.59 SW:P15944 (Vanderslice et al. (1989). Biochemistry28:4148-55) (48.2% identity in 274 aa. overlap). The next matchcorresponding to a known protein is human beta-tryptase precursor;tryptase 2 EC 3.4.21.59 SW:P20231 (Miller et al. (1990). J. Clin.Invest. 86:864-700) (44.3% identity in 273 aa. overlap). A phylogenetictree of an alignment of the deduced protease EOS amino acid sequencewith other members of the S1 serine protease family is shown in FIG. 2as determined using the MegAlign 3.1.7 program (DNASTAR Inc., Madison,Wis.).

EXAMPLE 2

Tissue Distribution of the Protease EOS mRNA

We employed a highly sensitive PCR profiling technique to identify thetissue distribution of protease EOS mRNA. For this application, severalhuman cDNA libraries (all were from Clontech, (Palo Alto, Calif.) exceptthe CHRF-288 megakaryocytic cell line and human gel filtered plateletlibraries which we constructed using the ZAP Express cDNA system(Stratagene, La Jolla, Calif.). The PCR primers for the profilinganalysis were as follows:

SEQ.ID.NO.:2: 5′-GAGAAAGTCAGATTCACAGC-3′

SEQ.ID.NO.:3: 5′-CTGCTTAGGGTCTCTTTAGG-3′

Briefly, the 50 □l PCR reactions used 1 □l of diluted phage stock (˜10⁸to 10¹⁰ pfu/ml) from each of the cDNA libraries tested. Reactions wereinitially denatured at 94° C. for 5 minutes and subjected to 35 cyclesof 94° C. for 20 seconds; 56° C. for 20 seconds; and then 72° C. for 30seconds followed by a final 72° C. elongation for 10 minutes A nestedprimer probe of the sequence SEQ.ID.NO.:4:5′-TGAGCGGCCTTTAAGAGTTGAGAGACAGCCGGCAGGGAAT-3 was radiolabeled usinggamma ³²P-ATP and T4 polynucleotide kinase (Life Technologies,Gaithersberg, Md.) and unincorporated label was removed, following thereaction, using a QIAquick nucleotide removal column (Qiagen, Valencia,Calif.). The ³²P end-labeled nested primer probe (1×10⁵ cpm) wascombined with 10 □l of each sample following the PCR reaction. The PCRproduct-probe mixtures were denatured at 94° C. for 5 minutes;hybridized at 60° C. for 15 minutes, and cooled to 4° C. The annealedsamples (10 □l ) were electrophoresed in 6% Tris-Borate-EDTAnon-denaturing polyacrylamide gels (Novex), dried and exposed byautoradiography. A PCR profile of the cDNA libraries used in FIG. 3 withbeta-actin PCR primers and labeled nested primer probe produced abeta-actin PCR product in all samples examined.

As seen in FIG. 3, the distribution of protease EOS mRNA is highlyrestricted to specific tissues and cell types. The tissue typesexpressing the protease EOS transcript are retina, ovary, and spleen,stomach, and to a lesser extent thymus, uterus and thalamus. Ofparticular significance is that EOS protease mRNA is not expressed inpancreas, liver or prostate, tissues normally found to express numerousserine protease genes. It is of interest that we detect the protease EOSPCR product in cDNA libraries constructed from human gel filteredplatelets. This strongly suggests that protease EOS is expressed inhuman platelets. The platelets used to construct the cDNA libraryanalyzed in the PCR tissue profile of FIG. 3 contained extremely lowlevels of contaminating erythrocytes and other blood cells (<1 per 10⁶platelets), so we can not rule out the possibility of leukocytecontamination, which would generate a false positive signal in thissensitive PCR assay. Cell localization by in situ hybridization orsensitive in situ PCR, and confirmation with protease EOS specificantisera, could be employed to address this issue.

EXAMPLE 3

Construct Generation for the Expression of Active Protease EOS

Since members of the S1 protease family are most often synthesized asinactive zymogen precursors, and require limited proteolysis to becomeproteolytically active, we have developed a zymogen activation constructto express and permit the generic activation of heterologous serineprotease cDNAs. This construct features a bovine preprolactin signalsequence fused in-frame with the MoAb M2 anti-FLAG antibody epitope aspreviously described (Ishii et al. (1993). J. Biol. Chem. 268:9780-6)for the purposes of secretion and antibody detection respectively (PF).Significantly, this construct also contains the enterokinase cleavagesite from human trypsinogen I (EK) fused in-frame and downstream fromthe signal sequence. At the C-terminus, preceding a stop codon, areadditional sequences encoding the hemagglutinin (HA) epitope and 6histidine (6XHIS) codons for detection with the anti-HA antibody MoAb 12CA5 (Boehringer Mannheim Corp., Indianapolis, Ind.) and affinitypurification on nickel resins respectively. A unique Xba I restrictionenzyme site, immediately upstream of the epitope/affinity tag sequenceand downstream of the PFEK prepro sequence described above, and is thepoint of in-frame insertion of the catalytic domain of a heterologousserine protease cDNA (FIG. 4). The zymogen activation vector describedabove has been cloned into a modified pFastBacl transplacement plasmidto generate PFEK-HA6XHIS-TAG FB.

The purified plasmid DNA of the full length protease EOS cDNA was usedas a template in a 100 □l preparative PCR reaction using theAdvantage-GC cDNA Polymerase Mix (Clontech, Palo Alto, Calif.) inaccordance with the manufacturer's recommendations. The primers used

SEQ.ID.NO.:5: EOS Xba-U 5′-GGGATCTAGAGGACGGAGAGTGGCCGTGGC-3′

SEQ.ID.NO.:7: EOS Xba-L 5′-CTCATCTAGAAGCATTAGAAGTGACGCGAGCCTG-3′

contained Xba I cleavable ends, and were designed to flank the catalyticdomain of the protease EOS and generate the protease EOS Xba I catalyticcassette. The preparative PCR reaction was run at 18 cycles of 94° C.for 30 seconds ; 68° C. for 2.5 minutes.

The preparative PCR product was phenol/CHCl₃ (1:1) extracted once, CHCl₃extracted, and then EtOH precipitated with glycogen (Boehringer MannheimCorp., Indianapolis, Ind.) and carrier. The precipitated pellet wasrinsed with 70% EtOH, dried by vacuum, and resuspended in 80 ul H₂O, 10ul restriction buffer number 2 and 1 ul 100× BSA (New England Biolabs,Beverly, Mass.). The product was digested for 3 hr. at 37° C. with 200units Xba I restriction enzyme (New England Biolabs, Beverly, Mass.).The Xba I digested product was phenol/CHCl₃ (1:1) extracted once, CHCl₃extracted, EtOH precipitated, rinsed with 70% EtOH, and dried by vacuum.For purification from contaminating template plasmid DNA, the productwas electrophoresed through 1.0% low melting temperature agarose (LifeTechnologies, Gaithersberg, Md.) gels in TAE buffer (40 mm Tris-Acetate,1 mM EDTA pH 8.3) and excised from the gel. An aliquot of the excisedproduct was then used for in-gel ligations with the Xba I digested,dephosphorylated and gel purified, zymogen activation vector describedabove. Clones containing the EOS Xba cassette, inserted in the correctorientation to generate the construct PFEK-protease EOS-HA6XHIS-TAG 64,were confirmed by sequence analyses to ensure that the propertranslational register with respect to the NH₂-terminal PFEK preprosequence and C-terminal HA6XHIS epitope/affinity tag was maintained.

EXAMPLE 4

Expression of Recombinant Protease EOS

The recombinant bacmid containing the PFEK-protease EOS-HA6XHISconstruct was prepared from bacterial transformation, selection, growth,purification and PCR confirmation in accordance with the manufacturer'srecommendations. Cultured Sf9 insect cells (ATCC CRL-1711) weretransfected with purified bacmid DNA and several days later, conditionedmedia containing recombinant PFEK-EOS-HA6XHIS baculovirus was collectedfor viral stock amplification. Sf9 cells growing in Sf-900 II SFM at adensity of 2×10⁶/ml were infected at a multiplicity of infection of 2 at27° C. for 80 hours, and cell pellets were collected for purification ofPFEK-EOS-HA6XHIS.

EXAMPLE 5

Purification, and Activation of Recombinant Protease EOS

Cells were lyzed on ice in 20 mM Tris (pH7.4), 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, leupeptin (1 □g/ml), andpepstatin (1 □g/ml). Cell lysates were mixed with anti-FLAG M2 affinitygel (Eastman Kodak Co., New Haven, Conn.) and bound at 4° C. for 3 hourswith gentle rotation. The zymogen-bound resin was washed 3 times withTBS buffer (50 mM Tris-HCl, 150 mM NaCl at a final pH of 7.5), andeluted by competition with FLAG peptide (100 □g/ml) in TBS buffer. Theeluted zymogen was dialyzed overnight against TBS in Spectra/Pormembrane (MWCO: 12,000-14,000) (Spectra Medical Industries, Inc.,Huston, Tex.). Ni-NTA (150 □l of a 50% slurry/per 100 □g of zymogen)(Qiagen, Valencia, Calif.) was added to 5 ml the dialyzed sample andmixed by shaking at 4° C. for 60 minutes The zymogen-bound resin waswashed 3 times with wash buffer [10 mM Tris-HCl (pH 8.0), 300 mM NaCl,and 15 mM imidazole], followed by with a 1.5 ml wash with ds H₂O.Zymogen cleavage was carried out by adding enterokinase (10 U per 50 □gof zymogen) (Novagen, Inc., Madison, Wis.; or Sigma, St. Louis, Mo.) tothe zymogen-bound Ni-NTA beads in a small volume at room temperatureovernight with gentle shaking in a buffer containing 20 mM Tris-HCl (pH7.4), 50 mM NaCl, and 2.0 mM CaCl₂. The resin was then washed twice with1.5 ml wash buffer. The activated protease EOS-HA6XHIS was eluted withelution buffer [20 mM Tris-HCl (pH 7.8), 250 mM NaCl, and 250 mMimidazole].

Eluted protein concentration was determined by a Micro BCA Kit (Pierce,Rockford, Ill.) using bovine serum albumin as a standard. Amidolyticactivities of the activated protease EOS-HA6XHIS was monitored byrelease of para-nitroaniline (pNA) from the synthetic substratesindicated in FIG. 6. The chromogenic substrates used in these studieswere all commercially available (Bachem California Inc., Torrance, Pa.;American Diagnostica Inc., Greenwich, Conn.; Kabi Pharmacia Hepar Inc.,Franklin, Ohio). Assay mixtures contained chromogenic substrates at 500uM and 10 mM Tris-HCl (pH 7.8), 25 mM NaCl, and 25 mM imidazole. Releaseof pNA was measured over 120 minutes at 37° C. on a micro-plate reader(Molecular Devices, Menlo Park, Calif.) with a 405 nm absorbance filter.The initial reaction rates (Vmax, mOD/min) were determined from plots ofabsorbance versus time using Softmax (Molecular Devices, Menlo Park,Calif.). The specific activities (nmole pNA produced /min/ug protein) ofthe activated protease EOS-HA6XHIS for the various substrates arepresented in FIG. 6. No measurable chromogenic amidolytic activity wasdetected with the purified unactivated PFEK-protease EOS-HA6XHISzymogen.

Electrophoresis and Western Blotting Detection of Recombinant ProteasesEOS

Samples of the purified PFEK-protease EOS-HA6XHIS zymogen or activatedprotease EOS-HA6XHIS, denatured in the presence of the reducing agentdithiothreitol (DTT), were analyzed by SDS-PAGE (Bio Rad, HerculesCalif.) stained with Coomassie Brilliant Blue. For Western blotting,gels were electrotransfer to Hybond ECL membranes (Amersham, ArlingtonHeights, Ill.). The FLAG-tagged PFEK-protease EOS-HA6XHIS zymogenexpressed from infected Sf9 cells was detected with anti-Flag M2antibody (Eastman Kodak Co., New Haven, Conn.). The secondary antibodywas a goat-anti-mouse IgG (H+L), horseradish peroxidase-linked F(ab′)2fragment, (Boehringer Mannheim Corp., Indianapolis, Ind.) and wasdetected by the ECL kit (Amersham, Arlington Heights, Ill.).

EXAMPLE 6

Chromogenic Assay

Amidolytic activities of the activated serine proteases are monitored byrelease of para-nitroaniline (pNA) from synthetic substrates that arecommercially available (Bachem California Inc., Torrance, Pa.; AmericanDiagnostica Inc., Greenwich, Conn.; Kabi Pharmacia Hepar Inc., Franklin,Ohio). Assay mixtures contain chromogenic substrates in 500 uM and 10 mMTRIS-HCl (pH 7.8), 25 mM NaCl, and 25 mM imidazole. Release of pNA ismeasured over 120 min at 37° C. on a micro-plate reader (MolecularDevices, Menlo Park, Calif.) with a 405 nm absorbance filter. Theinitial reaction rates (Vmax, mOD/min) are determined from plots ofabsorbance versus time using Softmax (Molecular Devices, Menlo Park,Calif.). Compounds that modulate a serine protease of the presentinvention are identified through screening for the acceleration, or morecommonly, the inhibition of the proteolytic activity. Although in thepresent case chromogenic activity is monitored by an increase inabsorbance, fluorogenic assays or other methods such as FRET to measureproteolytic activity as mentioned above, can be employed. Compounds aredissolved in an appropriate solvent, such as DMF, DMSO, methanol, anddiluted in water to a range of concentrations usually not exceeding 100uM and are typically tested, though not limited to, a concentration of1000-fold the concentration of protease. The compounds are then mixedwith the protein stock solution, prior to addition to the reactionmixture. Alternatively, the protein and compound solutions may be addedindependently to the reaction mixture, with the compound being addedeither prior to, or immediately after, the addition of the proteaseprotein.

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7 1 1613 DNA Homo sapiens 1 ccacgcgtcc gaccagagtc caagccctag gcagtgccacccttacccag cccagccttg 60 aagacagaat gagaggggtt tcctgtctcc aggtcctgctccttctggtg ctgggagctg 120 ctgggactca gggaaggaag tctgcagcct gcgggcagccccgcatgtcc agtcggatcg 180 ttgggggccg ggatggccgg gacggagagt ggccgtggcaggcgagcatc cagcatcctg 240 gggcacacgt gtgcgggggg tcgctcatcg ccccccagtgggtgctgaca gcggcgcact 300 gcttccccag gagggcactg ccagctgagt accgcgtgcgcctgggggcg ctgcgtctgg 360 gctccacctc gccccgcacg ctctcggtgc ccgtgcgacgggtgctgctg cccccggact 420 actccgagga cggggcccgc ggcgacctgg cactgctgcagctgcgtcgc ccggtgcccc 480 tgagcgctcg cgtccaaccc gtctgcctgc ccgtgcccggcgcccgcccg ccgcccggca 540 caccatgccg ggtcaccggc tggggcagcc tccgcccaggagtgcccctc ccagagtggc 600 gaccgctaca aggagtaagg gtgccgctgc tggactcgcgcacctgcgac ggcctctacc 660 acgtgggcgc ggacgtgccc caggctgagc gcattgtgctgcctgggagt ctgtgtgccg 720 gctaccccca gggccacaag gacgcctgcc agggtgattctgggggacct ctgacctgcc 780 tgcagtctgg gagctgggtc ctggtgggcg tggtgagctggggcaagggt tgtgccctgc 840 ccaaccgtcc aggggtctac accagtgtgg ccacatatagcccctggatt caggctcgcg 900 tcacttctaa tgctagccgg tgaggctgac ctggagccagctgctggggt ccctcagcct 960 cctggttcat ccaggcacct gcctataccc cacatcccttctgcctcgag gccaagatgc 1020 ctaaaaaagc taaaggccac cccacccccc acccaccaccttctggctcc tctcctcttt 1080 ggggatcacc agctctgact ccaccaaccc tcatccaggaatctgccatg agtcccaggg 1140 agtcacactc cccactccct tcctggcttg tatttacttttcttggccct ggccagggct 1200 gggcgcaagg cacgcagtga tgggcaaacc aattgctgcccatctggcct gtgtgcccat 1260 ctttttctgg agaaagtcag attcacagca tgacagagatttgacaccag ggagatcctc 1320 catagctggc tttgaggaca cggggaccac agccatgagcggcctctaag agctgagaga 1380 cagccggcag ggaatcggaa ccctcagacc cacagccgcaaggcactgga ttctggcagc 1440 accctgaagg agctgggaag taagttcttc cccagcctccagataagagc cccgccggcc 1500 aatcccttca tttcaaccta aagagaccct aagcagagaacctagctgag ccactcctga 1560 cctacaaagt tgtgacttaa taaatgtgtg ctttaagctgccaaaaaaaa aaa 1613 2 20 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 2 gagaaagtca gattcacagc 20 3 20 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide 3ctgcttaggg tctctttagg 20 4 40 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 4 tgagcggcct ttaagagttg agagacagccggcagggaat 40 5 30 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide 5 gggatctaga ggacggagag tggccgtggc 30 6 34 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide 6ctcatctaga agcattagaa gtgacgcgag cctg 34 7 284 PRT Homo sapiens 7 MetArg Gly Val Ser Cys Leu Gln Val Leu Leu Leu Leu Val Leu Gly 1 5 10 15Ala Ala Gly Thr Gln Gly Arg Lys Ser Ala Ala Cys Gly Gln Pro Arg 20 25 30Met Ser Ser Arg Ile Val Gly Gly Arg Asp Gly Arg Asp Gly Glu Trp 35 40 45Pro Trp Gln Ala Ser Ile Gln His Pro Gly Ala His Val Cys Gly Gly 50 55 60Ser Leu Ile Ala Pro Gln Trp Val Leu Thr Ala Ala His Cys Phe Pro 65 70 7580 Arg Arg Ala Leu Pro Ala Glu Tyr Arg Val Arg Leu Gly Ala Leu Arg 85 9095 Leu Gly Ser Thr Ser Pro Arg Thr Leu Ser Val Pro Val Arg Arg Val 100105 110 Leu Leu Pro Pro Asp Tyr Ser Glu Asp Gly Ala Arg Gly Asp Leu Ala115 120 125 Leu Leu Gln Leu Arg Arg Pro Val Pro Leu Ser Ala Arg Val GlnPro 130 135 140 Val Cys Leu Pro Val Pro Gly Ala Arg Pro Pro Pro Gly ThrPro Cys 145 150 155 160 Arg Val Thr Gly Trp Gly Ser Leu Arg Pro Gly ValPro Leu Pro Glu 165 170 175 Trp Arg Pro Leu Gln Gly Val Arg Val Pro LeuLeu Asp Ser Arg Thr 180 185 190 Cys Asp Gly Leu Tyr His Val Gly Ala AspVal Pro Gln Ala Glu Arg 195 200 205 Ile Val Leu Pro Gly Ser Leu Cys AlaGly Tyr Pro Gln Gly His Lys 210 215 220 Asp Ala Cys Gln Gly Asp Ser GlyGly Pro Leu Thr Cys Leu Gln Ser 225 230 235 240 Gly Ser Trp Val Leu ValGly Val Val Ser Trp Gly Lys Gly Cys Ala 245 250 255 Leu Pro Asn Arg ProGly Val Tyr Thr Ser Val Ala Thr Tyr Ser Pro 260 265 270 Trp Ile Gln AlaArg Val Thr Ser Asn Ala Ser Arg 275 280

What is claimed is:
 1. A monospecific antibody that immunologically binds with protease EOS protein having an amino acid sequence as set forth in SEQ. ID. NO.7.
 2. The antibody of claim 1, wherein the antibody blocks protease activity of the protein.
 3. The antibody of claim 1, wherein the antibody is a purified monoclonal antibody.
 4. The antibody of claim 1, wherein the antibody is purified from mammalian antiserum containing anitibodies that bind with said protease EOS protein.
 5. The antibody of claim 4, wherein the mammalian antiserum is from a mammal selected from the group consisting of mice, rats, guinea pigs, rabbits, and goats. 